Metalloproteases of the neprilysin family

ABSTRACT

In this paper, we describe RT-PCR strategies that allowed us to identify and clone members of the NEP-like family. Degenerate oligoncleotide primers corresponding to consensus sequences located on either side of the HEXXH consensus sequence for zincins were designed and used in RT-PCR with mouse and human testis cDNAs. DNA fragments with lengths expected from the sequence of this class of enzympes were obtained. These DNA fragments were cloned and sequenced. Using this PCR strategy and the PCR fragments as probes to screen cDNA libraries, three zincin-like peptidases were identified in addition of known members of the family. The cDNA sequences allowed to derive specific probes for Northern and in situ hybridization, and probe human chromosomes to localize the gene and establish potential links to genetic diseases. Furthermore, these cDNA sequences were used to produce recombinant fusion proteins in  Escherichia coli  in order to raise specific antibodies. Finally, the cDNA sequences were cloned in mammalian expression vectors and transfected in various mammalian cell lines to produce active recombinant enzymes suitable for testing specific inhibitors.

This application is the U.S. National Phase of International ApplicationPCT/CA00/00147, filed Feb. 11, 2000, published in English under PCTArticle 21(2), which designated the U.S. PCT/CA00/00147 claims priorityto Canadian Patent Application No. 2,260,376, filed Feb. 11, 1999. Theentire content of these applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Peptides are used by cells from yeast to mammals to elicit physiologicalresponses. The use of peptides as messengers usually involves thefollowing steps: 1) production and release of the peptide by a specificcell, 2) interaction of the peptide with a receptor on the surface ofthe target cell, and 3) degradation of the peptide to terminate itsaction. The first and last steps of this scheme require theparticipation of proteases/peptidases. There is increasing evidence thatmembrane-associated zinc-metallopeptidases play important roles in bothof these steps. Although activation of prohormone precursors intobioactive peptides is generally performed by proteases of the subtilisinfamily located in the Trans-Golgi Network or in secretory granules ofthe cell (for a review see: (Seidah and Chretien, 1995)) a few peptidesneed a final processing step. This step involves the action ofmembrane-associated zinc-metallopeptidases. Two cases are particularlywell documented: angiotensin-converting enzyme (ACE) which cleavesinactive angiotensin I into angiotensin II (Corvol and Williams, 1997)and endothelin-converting enzymes (ECEs) which cleave isoforms of bigendothelins into endothelins (Turner, 1997a). In addition to their rolein peptide activation, cell surface zinc-metallopeptidases have alsobeen implicated in the termination of the peptidergic signal by breakingdown the active peptides into inactive fragments. One of the best knownof these peptidases is probably Neutral Endopeptidase-24.11 (Neprilysin,NEP) that has been implicated in the physiological degradation ofseveral bioactive peptides (Kenny, 1993). Interestingly, NEP and theECEs show significant structural similarities and appear to be membersof a family of peptidases that also includes PEX, a newly discovered andnot yet characterized peptidase, and the KELL blood group protein(Turner and Tanzawa, 1997b). Because of their important role asregulators of bioactive peptide activity, these enzymes (morespecifically NEP and the ECEs) have been identified as putative targetsfor therapeutic intervention, similar to the way ACE inhibitors are usedto control blood pressure. The recent discovery of PEX, another memberof the family, which appears to be involved in phosphate homeostasis,raised the possibility that other yet unknown members might exist.

Members of the NEP-like family are type II membrane proteins consistingof three distinct domains: a short NH2-terminal cytosolic sequence, asingle transmembrane region, and a large extracellular or ectodomainresponsible for the catalytic activity of the enzyme. There arepotential N-glycosylation sites and cysteine residues that are involvedin disulfide bridges stabilizing the conformation of the active enzyme.These enzymes are metalloenzymes with a Zn atom in their active site. Assuch, they belong to the zincin family of peptidases which ischaracterized by the active site consensus sequence HEXXH (Hooper,1994), where the two histidine residues are zinc ligands. In members ofthe NEP-like family of peptidases, the third zinc ligand is a glutamicacid residue located on the carboxy-terminus side of the consensussequence. This characteristic puts them in the gluzincin sub-family(Hooper, 1994). The model enzyme for gluzincins is thermolysin (TLN) abacterial protease whose 3D structure has been determined by X-raycrystallography (Holmes and Matthews, 1982). The active site of NEP hasbeen extensively studied by site-directed mutagenesis and severalresidues involved in zinc binding (Devault et al., 1988b; Le Moual etal., 1991; Le Moual et al., 1994), catalysis (Devault et al., 1988a;Dion et al., 1993), or substrate binding (Vijayaraghavan et al., 1990;Beaumont et al., 1991; Dion et al., 1995; Marie-Claire et al., 1997)have been identified (for a recent review see Crine et al., 1997).

SUMMARY OF THE INVENTION

Here, we developed an RT-PCR strategy to look for other members of thisimportant family of peptidases. This strategy allowed the molecularcloning and characterization of three additional NEP-like (NL)metallopeptidases (called NL-1, NL-2 and NL-3). Knowledge obtainedthrough these studies allows the generation of reagents (nucleic acidprobes and primers, antibodies and active recombinant enzymes) forfurther biochemical characterization of these enzymes and their patternof expression and will greatly help the rational design of specificinhibitors that could be used as therapeutic agents.

Accordingly, the present invention relates to the following products:

-   A. Degenerate primers for screening new NEP-related enzymes;-   B. NL-1, NL-2 and NL-3 proteins as NEP-related enzymes;-   C. Nucleic acids encoding these enzymes;-   D. Antibodies directed against the enzymes;-   E. Recombinant vectors comprising the nucleic acids encoding the    enzymes and hosts transformed therewith;-   F. Fragments of the nucleic acids useful as probes or primers to    hybridize and detect the presence of an NL-1, NL-2 and NL-3 genes,    or to hybridize and amplify and produce gene fragments;-   G. Soluble forms of NL-1, NL-2 and NL-3; and-   H. Nucleic acids comprising the N-terminal part of NL-1 or NL-2    which terminates with a sequence encoding a furin recognition site,    such nucleic acids being useful for making a fusion protein with the    ectodomain of any protein of interest, and for releasing a soluble    form of that protein of interest (containing the ectodomain) in the    medium.

Also the present invention relates to the following methods:

-   A. A method for screening NEP-related enzymes that make use of    degenerate primers or probes selected from a region of NEP family    members in a highly conserved region, namely around the zinc-binding    sites; and-   B. A method for producing NL-1, NL-2 or NL-3 that includes the steps    of culturing the above recombinant host and recovering NL-1, NL-2    and NL-3 gene products therefrom.

The present invention will be described hereinbelow by referring tospecific embodiments and appended figures, which purpose is toillustrate the invention rather than to limit its scope.

In the first section, general procedures leading to the identificationand localization of NL-1, NL-2 and NL-3 are given. In the secondsection, slightly different procedures are given for completing orreiterating the work performed on NL-1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Amino acid sequence comparison of human NEP (SEQ ID NO: 1), PEX(SEQ ID NO: 2), KELL (SEQ ID NO: 3) and ECE1 (SEQ ID NO: 4) peptidases.Amino acid sequences in boxes are those used to design theoligonucleotide primers. Numbers and arrows under the sequences identifythe primer and its orientation.

FIG. 2: Sequences of the oligonucleotide primers used in the PCRreactions (1A: SEQ ID NO: 5; 1B: SEQ ID NO: 6; 2A: SEQ ID NO: 7; 2B: SEQID NO: 8; 3: SEQ ID NO: 9; 4: SEQ ID NO: 10; and 5: SEQ ID NO: 11).

FIG. 3: Nucleotide (SEQ ID NO: 12) and amino acid (SEQ ID NO: 13)sequence of the mouse NL-1 cDNA. The sequence of the DNA fragmentobtained by PCR is in brackets.

FIG. 4: Partial nucleotide (SEQ ID NO: 14) and amino acid sequence (SEQID NO: 15) of the human NL-2 cDNA. The sequence of the DNA fragmentobtained by PCR is in brackets.

FIG. 5: Partial nucleotide (SEQ ID NO: 16) and amino acid sequence (SEQID NO: 17) of the human NL-3 cDNA.

FIG. 6: Amino acid sequence comparison of NEP (SEQ ID NO: 1), NL-1 (SEQID NO: 13), NL-2 (SEQ ID NO: 15) and NL-3 (SEQ ID NO: 17) peptidases.

FIG. 7: In situ hybridization of mouse testis sections using NL-1 as aprobe.

FIG. 8: In situ hybridization of mouse sections using mouse NL-3 as aprobe.

FIG. 9: In situ hybridization of mouse spinal chord sections

FIG. 10: Expression of NL-1 in mammalian cells.

FIG. 11: Activity of recombinant soluble NL-1.

FIG. 12: Expression of a soluble form of NL-3 using NL-1 amino-terminaldomain.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Section 1)

Materials and Methods

DNA and RNA manipulations

All DNA manipulations and Northern blot analysis were performedaccording to standard protocols (Ausubel et al., 1988; Sambrook et al.,1989).

mRNA purification and cDNA synthesis

mRNAs were prepared from mouse testis using Quick Prep Micro mRNApurification kit (Pharmacia Biotech). Purified mRNAs were kept at −70°until ready used. First strand cDNA was synthesized from 1 μg of mRNAusing the First-Strand cDNA synthesis kit (Pharmacia Biotech). The humantestis cDNA library was obtained from Clonetech.

Polymerase chain reaction protocol

PCR was performed in a DNA thermal cycler with 5 μl of cDNA template and1 μl of Taq DNA polymerase in a final volume of 100 μl, containing 1 mMMgCl₂, 2 μM of each primer oligonucleotide, 20 μM of each dNTP and 5%DMSO. Cycling profiles included an initial denaturation step of 5 min at94° C., followed by 30 cycles of 1 min at 94° C., 1 min at 40° C. and1.5 min at 72° C. A final extension step was performed at 72° C. for 10min. The amplified DNA was loaded on a 2% agarose gel and visualized bystaining with ethidium bromide. Fragments ranging in size between500–700 bp were cut and eluted from the gel. If needed, a second roundof PCR was done with nested oligonucleotide primers, using 10 μl of thefirst PCR reaction, or of the eluted band cut from the agarose gel.Resulting fragments were ligated in pCR2.1 vector (Invitrogen) accordingto the distributor's recommendations. DH5α Escherichia coli cells weretransformed with the ligation mixture and grown on 2YT plates in thepresence of kanamycin. Plasmids were prepared from resistant cells andsequenced.

In situ hybridization on mouse tissues and chromosomal localization ofhuman genes

In situ hybridization on whole mouse slices or isolated tissues wasperformed as described previously (Ruchon et al., 1998).

To determine the chromosomal localization of human NL-2 and NL-3 genes,a technique for mapping genes directly to banded human chromosomes wasused. Metaphase chromosomes were obtained from lymphocytes cultured fromnormal human peripheral blood. Cells were synchronized with thymidineand treated with 5-bromodeoxyuridine (BrdU) during the last part of theS phase to produce R-banding. Biotin-labeling of the probe was done bynick-translation (Bionick, BRL) and the probe was visualized by indirectimmunofluorescence.

Antibody production

To raise antibodies against the new peptidases, the cDNA sequences ofeach protein was compared to that of other members of the family and thesequence segment showing the less homology was used. These sequences arefrom amino acid residues 273 to 354 for NL-1, from 75 to 209 for NL-2and from 143 to 465 for NL-3. These cDNA fragments were cloned in vectorpGEX2T (Pharmacia Biotechnology) downstream from and in phase withGluthatione-S-transferase (GST). Plasmids were transformed in E. colistrain AP401 and, induction of synthesis and purification of the fusionproteins were performed as recommended by the supplier. The NLpolypeptides were cleaved from the fusion protein with thrombin andpurified by SDS-PAGE. NL polypeptides were injected to rabbits or miceaccording to the following schedules: for rabbits, initial injection of150 μg of protein with boosts of the same amount 4 weeks and 8 weeksfollowing the initial injection; for mice, initial injection of 100 μgof protein followed by boosts of the same amounts 3 and 6 weeks later. Amonth after the last injection, sera were collected from the animals andtested by immunoblotting against the initial E. coli-produced antigensand the recombinant proteins produced in mammalian cell lines.

Production of monoclonal antibodies

cDNA fragments corresponding to amino acids segments of NLs selected toraise antibodies were used to construct a GST-fusion protein in E. coli.This fusion protein was purified from E. coli extracts by affinitychromatography on a glutathione-Sepharose column according to thesupplier's instructions (Amersham-Pharmacia). After thrombin cleavage,the NL portion of the GST fusion protein was further purified byelectroelution from a polyacrylamide gel. This material was used toimmunise 4 mice (5 injections of ≈50 μg of NL polypeptide). Blood wascollected from each mice after the immunisation schedule and thepresence of antibodies in mice serum was assessed by ELISA usingmicrotiter plates coated with NL polypeptide from E. coli extracts. Micesera were also tested for the presence of NL antibodies by Westernblotting extracts of mammalian cells transfected with the NL expressionvectors. One mouse selected for its high titer of NL specific antibodies(as measured by ELISA) was sacrificed and its spleen cells werecollected and immortalised by fusion with myeloma cells(strain:P3-X63Ag.653 from ATCC) as described previously (Crine 1985). Hybridomacells were selected for their ability to grow in HAT selection mediumand cloned by several rounds of limiting dilution. Hybridomas showingproper affinity and specificity to the enzymes NL-1, NL-2 and NL-3 whereselected.

Expression of NLs in cultured mammalian cells and enzymatic assays ThecDNAs for NL-1 and NL-3 were cloned in vectors pcDNA3 or pRcCMV(Invitrogen) and introduced by transfection in mammalian cell linesaccording to procedures already described in our laboratory (Devault etal., 1988a). Procedures to prepare extracts of cellular proteins orculture media were also described in previous papers (Devault et al.,1988a; Lemay et al., 1989). The presence of NLs in these extracts wasmonitored by immunoblotting using specific antibodies.

Extracts of cellular proteins and culture media were assayed forenzymatic activity. Two tests were performed. The first used[³H]-Tyr-(D)Ala₂-Leu-enkephalin as substrate and was performed accordingto Lemay et al., (1989). The second used bradykinin as substrate and wasperformed as described by Raut et al. (1999).

Results

Cloning of NL-1, a new member of the NEP family

The molecular cloning in the past few years of ECEs, PEX and KELL showedthat all these proteins have between 50 and 60% similarity with NEP.This observation led us to believe that these peptidases are part of anextended family and that there could be still additional members to bediscovered. To test this hypothesis, we aligned the amino acid sequencesof the members of the NEP-like family and designed degenerateoligonucleotide primers to be used in RT-PCR reactions (FIGS. 1 and 2).These primers were located on either side of the HEXXH consensussequence for zincins. Because they are highly degenerate, primers 1 and2 were each subdivided into two pools, 1A-1B, and 2A-2B, respectively(FIG. 2). Any PCR amplified DNA fragment that corresponds to a peptidaseof the family should normally contain the consensus sequence and beeasily recognized by sequencing of the cloned fragments. Using thisstrategy, we first performed PCR reactions with primer pairs 1A-3 and 1B-3. The amplified DNA migrates mostly as a smear starting at around 700bp and going down to 100 bp. As the expected fragments should be around550 bp, we isolated from the gel the section corresponding to DNAfragments longer than 500 bp. A second round of PCR reactions wasperformed with both crude PCR products of the first reaction andisolated DNA bands, using primers 2A-3 and 2B-3. The expected 296 bpfragment was seen on the gel (not shown).

Cloning of these DNA fragments generated approximately 350 clones, ofwhich 44 were sequenced. Nine of these had no inserts or corresponded tosequences not related to the NEP family, 24 corresponded to NEP, 3 toPEX, and 8 corresponded to one putative new member of the family, sincethey all contained the HEXXH consensus sequence for zincins and showed65% homology with mouse NEP (in boxes FIG. 3). This fragment was thenused to screen a mouse testis cDNA library, and allowed us to isolate acomplete cDNA of 2592 nucleotides (FIG. 3). The identity of thissequence with other members of the family is presented in Table I. Thisnew member was called NL-1, for NEP-like peptidase 1.

Cloning of NL-2 and NL-3

A strategy similar to that described for amplification of enzymes of theNEP family from mouse testis cDNAs was used with a human testis cDNAlibrary using two different oligonucleotide primers. This time, DNAfragments of approximately 900 bp were obtained and cloned. Ten cloneswere sequenced, revealing the presence of NEP and two new peptidases ofthe family that we have called NL-2 and NL-3.

The NL-2 PCR fragment was 879 nucleotides in length and encoded a 293amino acid residue segment probably located in the carboxy-terminaldomain of this putative peptidase (in brackets FIG. 4). This PCRfragment was then used to screen a lambda gt10 human brain cDNA library.It allowed the isolation of other cDNA fragments which overlap partiallywith the NL-2 PCR fragment. Fusion of these lambda clones and the PCRfragment resulted in an open reading frame of 770 amino acid residues.The use of 5′ RACE protocols with human testis cDNA libraries allowedcompletion of the sequence of NL-2 ORF (FIG. 4). This ORF codes for aputative protein that is about 80% identical to the mouse NL-1 protein(FIG. 6). Across species, members of the NEP, PEX, ECEs sub-familieshave highly conserved sequences (more than 94% identity). Although asequence identity of about 80% only exists between the novel humanprotein and mouse NL-1, these proteins share unique characteristics thatmake possible the fact that NL-2 protein may be the human homologue ofNL-1. The identity of NL-2 with other members of the family is presentedin Table I.

The 879 bp PCR fragment encoding NL-3 showed an open reading frame of293 amino acid residues (FIG. 5, in brackets). Sequence analysis of NL-3showed that it was 94.2% identical to an EST sequence from mouseembryonic tissue present in publicly accessible DNA data banks. Thismouse EST sequence, commercially available from American Tissue and CellCulture (ATCC), had been obtained previously by our laboratories.

Since Northern blot analysis of human tissues with the NL-3 PCR fragmentshowed the expression of this protein in spinal chord (see below), thesame PCR DNA fragment was used to screen by hybridization a human spinalchord cDNA library constructed in phage A vectors. One clone contained afull-length ORF of 752 amino acid residues that encompassed the 293amino acid residue ORF of the PCR fragment. Further probing, cloning andsequencing lead to the obtention of NL-3 full sequence, shown in FIG. 5.

FIG. 6 presents a comparison of the amino acid sequence of the newNEP-like enzymes and Table I shows the extent of identity betweenmembers of the family.

Cellular distribution of NL-1, NL-2 and NL-3 peptidases

Determining the tissue distribution of NL-1, NL-2 and NL-3 may provideclues to identify the peptidergic systems in which they are involved. Itwill be particularly interesting to compare the tissue distribution ofthese peptidases with that of NEP and the ECEs to determine whether ornot the physiological functions of NL-1 and/or NL-2 and/or NL-3 mayoverlap those of NEP and/or ECEs.

In situ hybridization (ISH), using our mouse cDNA, was used to determinethe spatial and temporal expression of NL-1 during mouse development, asdone previously for PEX (Ruchon et al., 1998)). Serial sections of wholefoetal (12, 15 and 19 dpc) and adult mice (1, 3 and 6 days old) werehybridized with an [³⁵S]-labeled RNA probe. FIG. 7 shows a section ofmouse testis which was the only tissue identified to express NL-1 bythis technique. Cells of seminiferous tubules are specifically labeledbut spermatids located near the center of the tubule showed strongestlabeling. These cells are in the last stage of maturation intospermatozoids. The presence of NL-1 in testis has now been confirmed byNorthern analysis of mouse tissues (see FIG. 10). Other tissues expressNL-1, when analyzed by RT-PCT, which is a more sensitive assay (notshown).

A similar approach was used to determine the localization of NL-3 usingthe mouse EST obtained from ATCC. FIG. 8 shows sections of whole mouseat 17 days of embryonic development and 4 days post-natal. Severaltissues are expressing this putative peptidase including brain, where itis associated with neurons (FIG. 9), spinal chord, liver, spleen andbones. Labeling was stronger in bones from Hyp mouse, an animal modelfor hypophosphatemic rickets (FIG. 8). In bones, NL-3 was found to beexpressed by osteoblasts (not shown).

Northern blotting experiments were performed on several tissues withNL-2 and NL-3 probes. A Human Multiple Tissues Northern Blot (Clontech)was hybridized with specific probes. A single RNA band of approximately4.0 kb was revealed by the probe for NL-2. Expression of NL-2 isrestricted to brain and spinal cord (not shown). However, RT-PCR hasshown the presence of this enzyme in testis (not shown).

A single RNA band of approximately 3.0 kb was detected with the specificprobe for NL-3 (not shown). NL-3 expression was observed mainly inovary, spinal cord and adrenal gland.

Chromosomal localisation of the human gene for NL-2 and NL-3

As a mean to get clues on the function of the new metallopeptidases invertebrates, we have localized the new cDNAs on human chromosomes, inorder to look for a possible link between the gene locus and mappedgenetic diseases in humans. To do so, we have mapped the NL-2 and NL-3genes by high-resolution fluorescence in situ hybridization (FISH). NL-2was localized to chromosome band 1p36. Consistent with the cellulardistribution of NL-2 in humans, genetic diseases of the CNS such asdyslexia, neural tube defect, neuroblastoma, neuronal type ofCharcot-Marie-Tooth disease have all been mapped in this region andrepresent potential targets for a role of NL-2 in humans. NL-3 waslocalized to chromosome band 2q37. Consistent with a role of NL-3 inbones, a form of Albright hereditary osteodystrophy was mapped to thesame chromosomal locus (Phelan et al., 1995).

In view of the foregoing, NL-2 and NL-3 are metallopeptidases that areassumed to be immediately useful as markers for a disease or disorderassociated with human chromosomal locus 1p36 and 2q37, respectively.Their localization on a chromosome band associated with known diseasessuggests that they may be expressed or co-expressed with one or moregenes, as a cause or a consequence of disease development. It ispossible that these enzymes are up or down regulated, alone or alongwith other genes involved in a disease. Therefore, antibodies or otherligands specific to NL-2 or NL-3 may be used for a diagnostic purpose,as well as primers or probes in diagnostic assays using nucleic acidhybridization or amplification techniques. Otherwise, primers or probesdirected against the nucleic acids of NL-2 and NL-3 would be useful tomap the mutations of a gene located in close proximity and involved inthe disease. Therefore, no matter which exact function NL-2 and NL-3gene products have, their chromosomic localization provides onediagnostic utility. This localization as well as the tissulardistribution provide information as to the disease and tissue to beinvestigated to elucidate the exact function of these enzymes.

NL-1 resembles NL-2, sharing with the latter about 80% homology in theamino sequence and sharing structural characteristics such as the furinrecognition sequence located at the proximal end of the ectodomain. NL-2might be the human homologue of mouse NL-1. If such was the case, thesetwo proteins would have a substantial degree of divergence and, maybe,different profiles of activity varying from one species to another.

Chromosomal localization of NL-1 was determined in mouse genome bySingle Strand Conformational Polymorphism (SSCP) in collaboration withThe Jackson Laboratory Backcross DNA Panel Mapping Resource. NL-1 waslocalized to the distal region of mouse chromosome 4 which correspondsto human chromosome region 1p36 where is located NL-2 gene. Thisreinforces our hypothesis that NL-1 and NL-2 are species variants.

Production of antibodies against NLs

Antisera collected from injected animals were first tested byimmunoblotting on GST-antigen fusion proteins produced in E. coli.Antiserum from one rabbit recognized the NL-1 -related polypeptide andantisera from one mouse and one rabbit reacted with the NL-3-relatedpolypeptide (results not shown). The anti NL-1 antiserum and the mouseanti NL-3 antiserum, which appeared more specific than the rabbitantiserum, were next tested by immunoblotting on extracts of proteinsand culture media from cells expressing NL-1 or NL-3 (see below).

Expression of NL-1 in CHO cells

The cDNA encoding the full-length NL-1 protein was cloned in themammalian expression vector pcDNA3-RSV and transfected in CHO cells.Stable cell lines were established by selection with the drug G418 andtested by immunoblotting for the presence of NL-1.

Small amounts of NL-1 were found in the extracts of transfected CHOcells (results not shown). This intracellular species was sensitive toendo H digestion, indicating that the sugar moiety was not mature andsuggesting ER localization (results not shown). The culture medium oftransfected CHO cells showed the presence of soluble NL-1 (FIG. 10).This extracellular species was resistant to endo H suggesting truetransport through the late secretory pathway. The cDNA sequence of NL-1predicts a type II transmembrane protein. The mechanism by which NL-1 istransformed into a soluble protein is not known presently. However,examination of the amino acid sequence revealed the presence of aputative furin cleavage site from residue 58 to 65 (FIG. 3). A similarsite is present in NL-2 sequence.

The soluble form of NL-1 was assayed for activity using[³H]-Tyr-(D)Ala₂-Leu-enkephalin and bradykinin as substrates. FIG. 11shows that NL-1 can degrade the enkephalin substrate (K_(m)=18±10 μM)and that this activity can be inhibited by phosphoramidon (IC₅₀=0.9±0.3nM) and thiorphan (K_(m)=47±12 nM), a general inhibitor of enzymes ofthe NEP family. Bradykinin is also a substrate for NL-1 (not shown).

Use of NL-1 amino-terminal domain to promote secretion

The observation that NL-1 ectodomain was secreted, possibly throughcleavage of the transmembrane segment by furin, raised the possibilityto promote secretion of exogenous proteins that could be spliced to NL-1amino-terminal domain (from initiator methionine to the furin site). Totest this hypothesis, the ectodomain of NL-3 (from the third cysteine tothe end) was spliced to NL-1 amino-terminal domain using a PCR strategyand the recombinant DNA cloned in expression vector pRcCMV. The fusionprotein was expressed by transfection of the vector in COS-1 and HEK 293cells. The culture media of transfected cells was analyzed byimmunoblotting using the mouse antiserum against NL-3. FIG. 12 shows thepresence of NL-3 in the spent culture media of both COS-1 and HEK 293cells. This result shows that NL-1 amino-terminal domain can be used topromote secretion of exogenous proteins.

The soluble form of NL-3 was assayed for activity using[³H]-Tyr-(D)Ala₂-Leu-enkephalin as substrate. No activity was found.

The previous experiment showed that it was possible to use theamino-terminal domain of NL-1 to promote secretion of an otherwisemembrane attached protein ectodomain. To verify whether the samestrategy could be used to promote secretion of small peptides, a PCRstrategy was used to splice human β-endorphin to the amino-terminaldomain of NL-1 and the recombinant DNA was cloned in vector pRcCMV. Thefusion protein was expressed by transfection of the vector in COS-1 andHEK 293 cells. The culture media of transfected cells was collected 48 hafter transfection and the peptides purified as described previously(Noël et al., 1989). The presence of β-endorphin in the extracts wasdetected by radioimmunoassay. The results showed that both COS-1 and HEK293 cells produced approximately 100 pg of β-endorphin per ml of culturemedium. Therefore, the N-terminus of LN-1 and NL-2 which ends with afurin-recognition site will be useful to produce the soluble form of aprotein of interest.

Section 2)

Materials and Methods

DNA manipulations

All DNA manipulations, phage library screening, and plasmid preparationswere performed according to standard protocols (Ausubel 1988; Sambrook1989). Site-directed mutagenesis was performed using a PCR-basedstrategy as described previously (Le Moual 1994).

mRNA purification and RT-PCR protocol for identification of new membersof the neprilysin family

mRNAs were prepared from mouse testis using Quick Prep Micro mRNApurification kit (Pharmacia Biotech). First strand cDNA was synthesizedfrom 1 μg of mRNA using the First-Strand cDNA synthesis kit (PharmaciaBiotech).

Two sense primers, oligonucleotide 3817(5′-TGGATGGAT/CGA/CIGG/AIACIA/CA-3′) and oligonucleotide 3719(5′-A/GTIGTITTT/CCCIGCIGGIA/GT/AIC/TTG/CCA-3′) correspondingrespectively to amino acid residues 459 to 465 and 552 to 560 of NEPsequence, and one antisense primer, oligonucleotide 3720(5′-AIICCICCIA/TC/TA/GTCIGCIG/AC/TA/GTTT/CTC-3′) corresponding to aminoacid residues 646 to 654 (see FIGS. 1 and 2), were synthesized. PCR wasperformed with 5 μl of cDNA template and 1 μl of Taq DNA polymerase in afinal volume of 100 μl, containing 1 mM MgCl₂, 2 μM of eacholigonucleotide 3817 and 3720, 200 μM of each dNTP and 5% DMSO. Cyclingprofiles included an initial denaturation step of 5 min at 94° C., 30cycles of 1 min at 94° C., 1 min at 40° C. and 1.5 min at 72° C., and afinal extension step at 72° C. for 10 min. One half of the amplified DNAwas fractionated on a 2% agarose gel and fragments ranging in sizebetween 500–700 bp were purified and resuspended in a final volume of 50μl. A second round of PCR was done with primers 3719 and 3720, using astemplate either 10 μl of the first PCR reaction or 5 μl of the purifiedfragments, and the new PCR products were ligated in pCR2.1 vector(Invitrogen). Several identical clones corresponded to a potential newmember of the NEP family. We called this member NL1 for NEP-like 1.

Cloning of full-length NL1 cDNA

The cloned NL1 PCR fragment was used as probe to screen a mouse testis λUni-ZAP™XR cDNA library (Stratagene). Twelve out of a hundred positivephages were plaque purified and subcloned into pBS SK vector(Stratagene). As the longest clone analyzed presented an incomplete ORF(pBS-NL1A), 5′RACE with primers located in vector(5′-TAGTGGATCCCCCGGGCTGCAG-3′, sense primer) and NL1(5′-ACCAAACCTTTCCTGTAGCTCC-3′, antisense primer, nt 1303 to 1324 of NL1;was subsequently performed on the DNA of the remaining semi-purifiedpositive clones. Amplification was performed with 1 μl of Ventpolymerase in a final volume of 100 μl containing 50 ng of DNA, 4 mM ofMgSO₄, 1 μM of each oligonucleotide, 200 μM of each dNTP and 10% DMSO.Cycling parameters included an initial denaturation step of 1 min at 94°C., 25 cycles of 30 sec at 94° C., 30 sec at 60° C. and 1 min at 72° C.,and an incubation of 10 min at 72° C. A PCR fragment of the expectedlength was subcloned into pCR2.1 vector (clone pCR-NL1A), but sequencingrevealed no initiator ATG codon. A nested 5′RACE was then performed onmouse testis cDNA using the Marathon Ready cDNA kit (Clontech) withsense oligonucleotides AP1 and AP2 (from the kit) and NL1 antisenseoligonucleotides 5′-CCTGAGGGCTCGTTTTACAACCGTCCT-3′ (nt 503 to 529 ofNL1) and 5′-CTCATCCCAGGAGAAGTGTAGCAGGCT-3′ (nt 475 to 502 of NL1) asrecommended by the supplier. The resulting fragment was cloned intopCR2.1 vector (pCR-NL1B). Since only ten bp were missing for theinitiator ATG codon, we reconstructed the 5′ end of the cDNA byPCR-amplifying clone pCR-NL1A with sense primer5′-CCACCATGGTGGAGAGAGCAGGCTGGTGTCGGAAGAAG-3′ (nt 332 to 364 of NL1; the10 missing nucleotides are underlined) and antisense primer5′-ACCAAACCTTTCCTGTAGCTCC-3′ (nt 1303 to 1324 of NL1) using Ventpolymerase as described above. The DNA fragment was then inserted intopCR2.1 (clone pCR-NL1C). The entire ORF was reconstituted followingdigestion of pBS-NL1A and pCR-NL1C with EcoRI and PflMI. The 5′ end ofNL1 cDNA was excised from pCR-NL1C and ligated into pBS-NL1A at thecorresponding sites, resulting in plasmid pBS-NL1B. For expressionstudies, a BamHlIApal fragment generated out of pBS-NL1B, correspondingto the full length cDNA of NL1, was inserted into pCDNA3/RSV [18]vector.

Production of polyclonal antibodies

A plasmid for the production in Escherichia coli of a GST fusion proteinwith NL1 was constructed using pGEX-4T-3 expression vector (PharmaciaBiotechnologies). A 255 bp fragment from NL1 was amplified by PCR withVent polymerase using sense primer5′-GCTACGGGATCCGTGGCCACTATGCTTAGGAA-3′ (nt 1139 to 1158) and antisenseprimer 5′-CGATTGCTCGAGTGGGMCAGCTCGACTTCCA-3′ (nt 1377 to 1396). BothpGEX-4T-3 and the PCR product were digested with BamHI and XhoI andligated. The recombinant protein was produced and purified according tothe supplier's instructions. Five weeks old female balb/c mice wereimmunized at monthly intervals for 3 months with 20 μg of therecombinant NL1 fragment in Freund's adjuvant and antisera weresubsequently collected.

Cell culture and transfection

Human Embryonic Kidney (HEK 293) cells were maintained in DMEM mediumcontaining 10% fetal bovine serum (FBS), and supplemented withpenicillin at 60 μg/ml, streptomycin at 100 μg/ml and fongizone at 0.25μg/ml. Transfections of cells with appropriate plasmids were performedby the calcium/phosphate-DNA co-precipitation method (Chang 1987). Toestablish permanent cell lines, G418 selection was initiated 48 h afterthe transfections at 400 −82 g/ml for 12 days and gradually decreased at100 μg/ml.

LLC-PK, cells transfected with pRcCMV-sNEP were maintained as describedpreviously (Lanctot 1995).

Immunoblot analysis

For immunoblot analysis, cells were incubated for 16 h in synthetic DMEMmedium containing 2 mM sodium butyrate. Cellular proteins weresolubilized as previously described (Dion 1995). Secreted proteinsrecovered in culture media were concentrated approximately 10 fold byultrafiltration. Immunoblot analysis were performed using the NENRenaissance kit with the polyclonal antibody specific to NL1 or theα1-antitrypsin inhibitor antibody (Calbiochem; LaJolla, Calif.) followedby the appropriate horseradish peroxidase-conjugated IgG (VectorLaboratories).

For the glycosylation studies, proteins were incubated withendoglycosidase H (endoH) or peptide:N-glycosidase (PNGaseF) assuggested by the distributor (NEB).

Enzymatic activity assays

NL1 activity was monitored and compared to sNEP activity using(Tyrosyl-[3,5-³H])(D-Ala₂)-Leu₅-enkephalin (50 Ci/mmol) (ResearchProducts International Inc.), as already described (Dion 1995; Devault1988). K_(m) values were determined by the isotope-dilution method. Theinhibitory effects of phosphoramidon and thiorphan were also assessed aspreviously described (Dion 1995).

HPLC analysis of the hydrolysis of Leu-enkephalin

Five μg of Leu₅-enkephalin were incubated at 37° C. for one hour in 50mM MES, pH 6.5, with concentrated culture medium of HEK 293 cellsexpressing NL1 (˜300 μg of total proteins) or LLC-PK₁ cells expressingsNEP (˜30 μg of total proteins), in absence or presence of 0.1 mMphosphoramidon. Hydrolysis products were separated by reversed-phaseHPLC as described previously [23]. Tyr-Gly-Gly and Phe-Leu were bothidentified by elution profiles of synthetic marker peptides.

Northern blot analysis

A mouse multiple tissue poly(A)⁺ mRNA blot (Clontech) was hybridizedwith a [³²P]dCTP random primer labelled probe in ExpressHyb solution(Clontech). The blot was washed according to the manufacturer'srecommendations and exposed to Fuji RX film for 7 days at −80° C. withintensifying screens.

RT-PCR screening of mouse tissues

First strand cDNA synthesis was performed with 1 μg of total RNA frommouse tissues and oligo(dT) as primer, using Gene Amp RNA PCR Core Kit(Perkin Elmer). For the PCR reactions, primers5′-TGGCGAGAGTGTGTCAGCTATGTC-3′ and 5′-CTTCCAAAATGTAGTCAGGGTAGCCAATC-3′were used with Taq polymerase. One tenth of the PCR products werevisualized on a 4% agarose gel.

In situ hybridization

To construct a plasmid for the synthesis of cRNA probes for ISH,pCR-NL1A was used as template to amplify a 452 bp fragment by PCR withsense primer 5′-GGAGCCATAGTGACTCTGGGTGTC-3′ (nt 416 to 439) andantisense primer 5′-GACGCTCAGCAGGGGCTCAGAGTC-3′ (nt 842 to 865). Theamplification product was inserted into pCRII vector (Invitrogen).Synthesis of riboprobes and protocols for ISH were as describedpreviously (Ruchon 1998).

Results

Cloning and sequence analysis of mouse NL1 cDNA

In order to isolate cDNAs for new members of the NEP family, wedeveloped an RT-PCR strategy based on fact that NEP, ECE-1 and PHEXshare regions of significant sequence identity. Following RT-PCR ontestis mRNAs with nested primers, a DNA fragment of approximately 300 bpwas amplified. This DNA fragment was cloned and the plasmids from 24independent colonies were sequenced: 3 clones had no insert, 4 cloneshad DNA fragments not related to the NEP family, 7 clones had sequencescorresponding to mouse NEP and 3 clones had sequences corresponding tomouse PHEX, showing that our approach efficiently allowed theidentification of members of the family. Moreover, 7 identical cloneshad a new cDNA presenting sequence similarities to members of the NEPfamily. The full-length cDNA was subsequently obtained by screening amouse testis A cDNA library followed by 5′RACE, as described underMaterials and Methods. Its nucleotide and deduced amino acid sequencesconfirm that we cloned a novel NEP-like protein, referred to thereafteras NL1.

NL1 cDNA spans 2925 nt, including a 5′-untranslated region of 331 nt, anopen reading frame of 2295 nt from nt 332 to nt 2626, and a3′-untranslated region of 299 nt. The sequence surrounding the proposedinitiator ATG conforms to the Kozak consensus (Kozak 1986). The deducedamino acid sequence of NL1 reveals a putative type 11 transmembraneprotein of 765 amino acid residues encompassing a short N-terminalcytoplasmic tail, a unique putative transmembrane domain, and a largeC-terminal extracellular domain. The ectodomain contains nine potentialN-glycosylation sites (Asn-X-Ser/Thr) and ten cysteine residuescorresponding to those conserved among all the members of the family,which are presumably involved in proper folding and in maintenance ofthe protein in an active conformation. All amino acid residues known tobe part of the active site of NEP are present in NL1. The predictedprotein presents greater similarities to NEP than to any other member ofthe family. Although NL1 shares numerous features with proteins of theneprilysin family, a notable aspect distinguishes it from the others:the first conserved cysteine residue of the ectodomain is more distant(34 amino acid residues) from the predicted transmembrane domain in NL1than it is in NEP (9 residues) or any other members of the family.Moreover, we noticed a putative furin cleavage site(-Arg₅₈-Thr-Val-Val-Lys-Arg₆₃-) between the end of the transmembranedomain and the first cysteine. This observation suggests that NL1 couldexist as a secreted rather than a membrane-bound protein.

NL1 expression in HEK 293 cells

HEK 293 cells were transfected with pCDNA3/RSV expression vectorcontaining NL1 cDNA, and a permanent cell line was established asdescribed under Materials and Methods (HEK/NL1 cells). Immunoblottingwith a polyclonal antibody showed that after 16 h of culture, most NL1was present in the culture medium with small amounts of the enzyme inthe cell extract. Secreted and cell-associated NL1 had apparentmolecular masses of approximately 125 and 110 kDa, respectively. Tocharacterize the glycosylation state of NL1, we next submitted therecombinant protein to deglycosylation by peptide:N-glycosidase F(PNGase F) and endoglycosidase H (endo H). PNGase F removes high mannoseas well as most complex N-linked oligosaccharides added in the Golgicomplex. In contrast, endo H removes N-linked oligosaccharide sidechains of the high mannose type found on proteins in the RER but whichhave not yet transited through the Golgi complex; thus, resistance toendo H can be used as an indication that the protein has traveledthrough the Golgi complex. PNGase F treatment showed that thecell-associated and secreted NL1 were N-glycosylated as theirelectrophoretic mobility increased following digestion. However, thesecreted NL1 migrated as a doublet after PNGase F treatment, with oneband co-migrating with cell-associated form and the second having aslower rate of migration. Since untreated and endo H-digested secretedNL1 are seen as single bands by SDS-PAGE, our observation suggests thata proportion of secreted NL1 undergoes further post-RER postranslationalmodification that renders some of the N-linked oligosaccharidesresistant to PNGase F digestion.

In contrast to secreted NL1, NL1 from cell extract was sensitive to endoH treatment. This result shows differences in the glycosylation state ofthe two species and suggests that the cell-associated form observed intransfected cells is an intracellular species that has not traveledthrough the Golgi complex.

Processing of NL1 by a subtilisin-like convertase

To determine whether a member of the mammalian subtilisine-likeconvertase family is responsible for NL1 presence in the culture mediumof transfected cells, we co-transfected transiently HEK 293 cells with aconstant amount of plasmid pCDNA3/RSV/NL1 and increasing amounts ofplasmid pCDNA3/CMV/PDX (Benjannet 1997). This latter vector promotes theexpression of the α1-antitrypsin Portland variant, α1-PDX, a knowninhibitor of subtilisin-like convertases (Anderson 1993). Immunoblotanalysis of the culture media of cells expressing both NL1 and α1-PDXindicated that NL1 secretion was strongly inhibited by the presence ofα1-PDX: a relation was observed between the amounts of α1-PDX and thelevel of inhibition of NL1 secretion.

To confirm that proteolysis by the subtilisin-like convertase occurredat the putative furin cleavage site identified in NL1 ectodomain(-Arg₅₈-Thr-Val-Val-Lys-Arg₆₃-), the amino acid residues Asn₆₂-Gly₆₃were substituted for Lys₆₂-Arg₆₃ by site-directed mutagenesis in vectorpCDNA3/RSV/NL1 and the mutated vector used to establish HEK 293 cellsexpressing the mutant protein (HEK/NL1mut cells). Immunoblot analysis ofthe culture media of HEK/NL1 mut cells showed that the mutation totallyabolished secretion of NL1. Furthermore, an additional form of NL1 witha molecular mass of 127 kDa was detected in the extract of these cells.This new species was resistant to endo H digestion and was foundassociated with membranes when HEK/NL1mut cells were fractionatedaccording to Chidiac et al. 1996 (result not shown).

NL1 enzymatic activity

Culture media from HEK 293 and HEK/NL1 cells were tested for enzymaticactivity using as substrate (Tyrosyl-[3,5-³H](D-Ala₂)-Leu₅-enkephalin, aknown NEP substrate. Activity was detected in the culture medium ofHEK/NL1 cells but not in that of HEK 293 cells. This activity increasedlinearly with the amounts of NL1 and with the incubation period,indicating that degradation of the substrate was due to NL1 enzymaticactivity.

We next characterized NL1 enzymatic parameters using the same substrateand compared them to those of an engineered soluble form of NEP (sNEP)(Lemay 1989). NL1 affinity for D-Ala₂-Leu₅-enkephalin was slightlyhigher than that of sNEP as shown by their K_(m) values of 18 μM and 73μM, respectively. Inhibition assays showed that phosphoramidon hadsimilar effects on NL1 and sNEP activity, with IC₅₀ values of 0.9 nM and0.5 nM respectively, and that thiorphan, a specific inhibitor of NEP,inhibited NL1 with an IC₅₀ of 47 nM, as compared with an IC₅₀ of 8 nMfor NEP.

Very low levels of phosphoramidon-sensitive activity was detected inextracts of HEK/NL1 cells (data not shown) consistent with the smallamounts of NL1 observed by immunoblotting.

To determine whether NL1 had cleavage site specificity similar to NEP,we incubated Leu₅-enkephalin in the presence of NL1 recovered from themedium of HEK/NL1 cells or in the presence of sNEP, and analyzed thedegradation products by RP-HPLC. Peaks co-migrating with standardTyr-Gly-Gly and Phe-Leu peptides were observed in both RP-HPLC profiles,indicating that both enzymes cleaved the substrate at the Gly₃-Phe₄peptide bond. This enkephalin-degrading activity was completelyinhibited by phosphoramidon (1 μM).

Tissue and cellular distribution of NL1 mRNA

Tissue distribution of NL1 mRNA was determined by Northern blot analysiswith a specific probe corresponding to the 5′end of the coding region ofNL1 cDNA. A single transcript of 3.4 kb was detected exclusively intestis among all the mouse tissues tested. Mouse tissues were alsoscreened by RT-PCR. Using this more sensitive technique, expression ofNL1 was observed in several other tissues including heart, brain,spleen, lungs, liver and kidney. Consistent with the Northern blotresults, RT-PCR analysis, although not strictly quantitative, detectedmore NL1 mRNA in testis than in other tissues.

To gain more insight into NL1 mRNA distribution, we examined by ISHcryostat sagital sections from a 4-day newborn mouse, as well assections from a 16-day old animal (p16) and adult tissues (heart, brain,spleen, lungs, liver, kidney and testis). The presence of NL1 mRNA wasdetected only in adult testis. Only the germinal cells in the luminalface of the seminiferous tubules were labeled. These cells wereidentified as round and elongated spermatids in all spermiogenesismaturational stages. Neither spermatozoa nor spermatocytes,spermatogonies or Sertoli cells were labeled. Interstitial cells werealso negative. Controls were performed with sense riboprobes, whichproduced only nonspecific background (data not shown). The 4-day oldmouse sagital sections and all other tissues tested were negative.

Discussion

The great interest in members of the Neprilysin family as putativetherapeutic targets, and the recent discovery of new members of thisimportant family of peptidases led us to investigate whether additionalmembers of the family remained to be identified. Using a PCR-basedstrategy, we cloned, from mouse testis, a partial cDNA encoding a newNEP-like enzyme that we called NL1. Analysis of the amino acid sequenceencoded by the full-length NL1 cDNA revealed that this member of thefamily resembles NEP the most: 55% identity and 74% similarity.Recently, the primary structure of a new zinc metallopeptidase fromtotal mouse embryo was reported (Ikeda 1999). This enzyme, called SEP,is found either as a soluble or a cell-associated form due toalternative splicing. NL1 shows only 3 amino acid differences with thesoluble form of SEP indicating that secreted SEP and NL1 are the sameenzyme. Our cloning strategy did not allow characterization of thecell-associated form of NL1 which is a minor species in mouse testis(Ikeda 1999).

The amino acid sequence of NL1 predicts a topology of a type II integralmembrane glycoprotein that is similar to the other members of thefamily. Treatment of the recombinant protein with PNGase F showed thatindeed NL1 possesses N-linked carbohydrate side chains. However, it isnot possible to determine precisely whether all nine putativeN-glycosylation sites are used, but the 30 kDa decrease in molecularmass upon PNGase F treatment suggests that most are glycosylated. It hasalready been shown that all asparagine residues in a Asn-X-Ser/Thrconsensus are glycosylated in rabbit NEP expressed in COS-1 cells andthat sugar moieties increase the stability and enzymatic activity of theprotein and facilitate its intracellular transport (Lafrance 1994).Three of NEP glycosylated Asn residues (Asn 145, Asn 285 and Asn 628)are conserved in NL1 (Asn 163, Asn 303 and Asn 643). Amongst theseresidues, Asn 145 and Asn 628 have been reported to influence NEPenzymatic activity (Lafrance 1994). In the same work, it has also beenshown that the effect of sugar addition on folding and intracellulartransport of NEP is a cumulative effect of all glycosylation sitesrather than a contribution of any particular one. Glycosylation of NL1may share similarities with that of NEP since we found their primarystructures and enzymatic activities to be very similar.

Surprisingly, expression of the cDNA by transfection of HEK 293 cellsshowed that most of the enzyme was secreted in the culture medium. Thesmall amount of NL1 associated with the cells was endo H-sensitive,suggesting that the cell-associated enzyme is a species that has not yetleft the RER. The presence of a furin cleavage site in NL1 sequencebetween the predicted transmembrane domain and the first conservedcysteine residue of the ectodomain led us to believe that a member ofthe mammalian subtilisin-like family of convertases was responsible forthe presence of NL1 in the culture medium. These enzymes are involved inprocessing a variety of precursor proteins such as growth factors andhormones, receptors, plasma proteins, matrix metalloproteinases,metalloproteases-desintegrins and viral envelope glycoproteins [for areview see: (Nakayama 1997). Site-directed mutagenesis of the furincleavage site (-Arg₅₈-Thr-Val-Val-Lys-Arg₆₃-) and expression of α1-PDX,a potent inhibitor of mammalian subtilisin-like convertases (Anderson1993), confirmed that a member of this family of endoproteases wasinvolved in NL1 secretion presumably by cleaving in carboxy-terminus ofArg₆₃. There are only a few examples of proteins which are processedfrom a membrane-bound precursor to a secreted form following cleavage bysubtilisin-like convertases; these include meprin and collagen XVII(Milhiet 1995; Schacke 1998). Three members of the subtilisin-likefamily of convertases, namely furin, PC4 and PC7, are known to beexpressed in germ cells (Nakayama 1992; Torri 1993; Seidah 1992, 1996).Whether one of these convertases generates secreted NL1 from itsmembrane form is under current investigation. In any case, NL1 is theonly known member of the neprilysin family that is secreted. This uniquefeature suggests that NL1 plays its physiological role in a contextdifferent from that of the membrane-bound peptidases, therebydiversifying the role of the peptidases of the neprilysin family. It isof interest that circulating forms of NEP in blood and urine have beendescribed, but they have generally been related to pathological orstressful conditions (Almenoff 1984; Deschodt-Lanckmann 1989; Johnson1985; Soleilhac 1996; Aviv 1995).

We have observed in cells expressing NL1 mutated at the furin cleavagesite the appearance of a species resistant to digestion by endo H. Thismutated protein was associated with cellular membranes. Taken together,these results indicate that NL1 is first synthesized and inserted in theRER membrane as a type II transmembrane protein. During intracellulartransport, NL1 is converted to a soluble form by the action of a memberof the mammalian subtilisin-like convertases. The identity of thecellular compartment where this process occurs is not known. However,mammalian subtilisin-like convertases are usually active in post-Golgicompartments of the secretory pathway suggesting that processing of NL1from the membrane bound form to the soluble form is a post-Golgi event.

Despite almost total abrogation of NL1 secretion, we observed only aslight accumulation of endo H-resistant NL1 in cells eitherco-expressing α1-PDX and NL1 (result not shown) or expressing mutatedNL1. This observation suggests that unprocessed NL1 is rapidly degraded.A similar behavior was reported for the Notch1 receptor expressed in thefurin-deficient cell line LoVo (Logeat 1998). The mechanism(s) by whichthese unprocessed proteins are degraded is still unknown. It isinteresting to point out that the spliceoform of SEP that has lost a 23amino acid peptide, including the furin cleavage site, generates acell-associated endo H-sensitive molecule (Ikeda 1999).

The most important observation regarding the NL1 primary structure isthe conservation of residues which in NEP are essential for catalysisand binding of substrates or inhibitors. This finding suggests that NL1could effectively act as an endopeptidase with a catalytic mechanismsimilar to that of NEP. This hypothesis was supported by thedemonstration that D-Ala₂-Leu₅-enkephalin, a peptide substrate oftenused to monitor NEP activity, was also an excellent NL1 substrate. Theaffinity of NL1 for D-Ala₂-Leu₅-enkephalin was even higher than that ofNEP, as reflected by a K_(m) value 4- to 5-fold lower. Furthermore, twowell known NEP inhibitors, phosphoramidon and thiorphan, also abolishedNL1 activity. Phosphoramidon, which inhibits NEP as well as ECE-1activity, albeit to a lesser extent (Turner 1996), had very similareffects on NL1 and NEP, with an IC₅₀ value for NL1 varying not more thantwo-fold from the value determined for NEP. Thiorphan, considered to bea more specific inhibitor of NEP, also inhibited NL1 activity, with anIC₅₀ six-fold greater than that for NEP. These results suggest that theactive sites of NL1 and NEP are similar. This hypothesis is supported bythe observation that secreted SEP degraded a set of peptides known to beNEP substrates, including substance P, bradykinin and atrial natiureticpeptide (Ikeda 1999). Taken together, these results illustrate theimportance of identifying and characterizing other member of the familyfor the design of highly specific inhibitors.

In agreement with the enzymatic parameters demonstrating that NL1 andNEP have similar catalytic sites, we have observed that both enzymescleaved Leu₅-enkephalin at the same peptide bond. This result suggeststhat NL1 hydrolyzes peptide bonds on the amino side of hydrophobic aminoacid residues as does NEP (Turner 1985). However, several other peptideswill have to be tested to confirm this specificity and to determinewhether NL1 has dipeptidyl carboxypeptidase activity as was shown forNEP (Malfroy 1982; Bateman 1989; Beaumont 1991) and more recently forECE-1 (Johnson 1999).

RT-PCR experiments with specific primers for the soluble andcell-associated forms of SEP showed a wide tissue distribution of theenzyme with the soluble form of SEP being predominant in testis and thecell-associated form in other tissues (Ikeda 1999). Our RT-PCR resultsconfirmed the wide tissue distribution of NL1. However, Northernblotting and in situ hybridization experiments indicated that expressionof NL1 is predominant in germ cells of mature testis. Interestingly,proenkephalin mRNA has been shown to be expressed in germ cells andsomatic cells of the testis (Torii 1993, Seidah 1992; Kew 1989; Mehta1994; Kilpatrick 1986, 1987). Specific functions for testicularenkephalin peptides have not yet been defined, but it is believed thatthey could act as intratesticular paracrine/autocrine factors. Inaddition to their putative role as mediators of testicular cellcommunication, it has also been demonstrated that proenkephalin productssynthesized by spermatogenic cells during spermatogenesis are stored inthe acrosome of human, hamster, rat and sheep spermatozoa and arerelease from sperm following acrosomal reaction (Kew 1990). It has thusbeen proposed that proenkephalin products may act as sperm acrosomalfactors during the fertilization process as well as intratesticularregulators secreted by spermatogenic cells. Since Leu₅-enkephalin wasfound to be a good substrate for NL1, opioid peptides originating fromproenkephalin could serve as physiological substrate for this newenzyme. In this way, NL1 would serve to regulate the activity of thesebioactive peptides.

Testis is the only tissue where the soluble form of SEP is predominant(Ikeda, 1999), suggesting a testis-specific alternative splicing.Expression of testis-specific molecular species of peptidases orprohormones, arising through diverse mechanisms, has been documented inthe past (Howard 1990; Jeannotte 1987). However, the physiologicalsignificance of these testis-specific species is not always clear. Inthe case of NL1 or SEP, it might allow local constitutive secretion bygerminal cells of an otherwise cell-associated enzyme, to regulatespermatogenesis much like several other proteolytic enzymes of theseminiferous tubules (Monsees 1998). Alternatively, it might allowaccumulation in acrosome with proenkephalin peptides and release uponacrosomal reaction. More exhaustive studies concerning NL1 localizationand physiological substrate identification will be needed to understandits role in the testis and possibly in the fertilization process.

Cloning of other members of the family

To find other members of the NEP-like family, we will use the sameRT-PCR strategy to amplify mRNA isolated from tissues known to beregulated by peptidergic systems (brain, thymus, kidney, heart, lung,ovary, pancreas, bone, bone marrow and lymphoid cells). In fact, many ofthese tissues are known to express at least one member of the familyand/or to control a peptidergic pathway on which peptidase inhibitorshave major effects. Amplified fragments will be cloned and the resultingclones will be sequenced and compared to the sequence of knownpeptidases, as described above. Pairs of degenerate primers in otherhighly conserved regions will also be designed to increase thepossibility of cloning other relevant peptidases.

DISCUSSION

As discussed above, peptidases of the NEP family known to date haveoften been found to play important physiological roles. This iscertainly true for NEP itself, ECEs and PEX, (see review above). Forthis reason, some of these enzymes (as it was the case for NEP and ECEin the past) might be interesting targets for the design of inhibitorsthat in turn could be used as therapeutic agents in various pathologicalconditions. However, it is of some concern that inhibitors designed forone enzyme may also inhibit to some extent other members of the family.This lack of specificity for an inhibitor used as a therapeutic agent inthe long term treatments such as those used as antihypertensive agentsfor instance, may cause unforeseen problems due to unwanted sideeffects. The objectives of the present work was to develop a strategy toclone new members of the NEP family of peptidases. The results presentedin this report clearly show that our strategy can be successful. We havedetermined the complete or partial nucleotide sequence of three cDNAsencoding putative enzymes of the NEP family.

These cDNA sequences are valuable tools and may be used to:

Produce antibodies

-   -   As shown in the present work, knowledge of NL cDNA sequences can        be used to raise specific antibodies. For example but not        exclusively, regions of less homology between the peptidases        (amino acid residues 50 to 450) can be used to synthesize        peptides whose sequences are deduced from the translation of the        cDNAs, and/or bacterially-expressed fragments of the cDNAs fused        for example but not exclusively to GST may be purified and        injected into rabbits or mice for polyclonal or monoclonal        antibody production. These antibodies can be used to:        -   identify by immunohistochemistry the peptidergic pathways in            which the peptidases are functioning;        -   study the physiopathology of NL-enzymes by immunoblotting or            immunohistochemistry on samples of biological fluids or            biopsies;        -   set up high through put screening assays to identify            NL-enzymes inhibitors. This can be done for example but not            exclusively by using the antibodies to attach the NL-enzymes            to a solid support;        -   purify NL-enzymes with said antibodies by            immunoprecipitation or affinity chromatography by            identifying antibodies capable of selectively binding to the            NL-enzymes in one set of conditions and releasing it in            another set of conditions typically involving a large pH or            salt concentration change without denaturing the NL-enzyme;        -   identify antibodies that block NL-enzymes activities and use            them as therapeutic agents. Blocking antibodies can be            identified by adding antisera or ascite fluid to an in vitro            enzymatic assay and looking for inhibition of NL-enzymes            activities. Blocking antibodies could then be injected to            normal or disease model animals to test for in vivo effects.

Derive specific RNA or DNA probes

-   -   As shown in the present work, knowledge of the nucleotide        sequence of the members of the NEP-family allows nucleotide        sequence comparisons and facilitate the design of specific RNA        or DNA probes by methods such as but not exclusively molecular        cloning, in vitro transcription, PCR or DNA synthesis. The        probes thus obtained can be used to:        -   derive specific probes or oligonucleotides for RNA and DNA            analysis, such as Northern blot and in situ hybridization,            chromosome mapping or PCR testing. These probes could be            used for genetic testing of normal or pathological samples            of biological fluids or biopsies;        -   make vectors for gene knock-out or knock-in in mice. The            long range PCR technique and/or screening of a mouse genomic            library with probes derived from the 5′-end of the cDNAs can            be used to isolate large exon/intron regions. We will then            substitute one or more of the cloned genomic DNA exons for            the neomycin resistance expression cassette for producing            homologous recombination and knock-out mice. Alternatively,            cDNAs coding for NLs will be used to overexpressed each of            these enzymes in transgenic mice. The cDNAs will be cloned            downstream from a promoter sequence, and injected in            fertilised mouse eggs. Depending on specific questions to be            answered, the chosen promoter sequence will allow expression            of the peptidases either in every tissues or in a cell- or            tissue-specific manner. Injected eggs will be transferred            into foster mothers and the resulting mice analysed for            peptidase expression;        -   replace defective NL genes in a gene therapy strategy. The            NL full length cDNAs could be cloned under the control of a            constitutive or inducible promoter having a narrow or wide            range of tissue expression and introduced with appropriate            vectors in subjects having defective genes;        -   synthesise oligonucleotides that could be used to interfere            with the expression of the NLs. For example but not            exclusively, oligonucleotides with antisens or ribozyme            activity could be developed. These oligonucleotides could be            introduced in subjects as described above;        -   isolate other members of the family. Screening cDNA and/or            genomic libraries with these cDNA probes at low stringency            may allow to clone new members of the NEP-like family.            Alternatively, alignment of the sequences may allow one to            design specific degenerate oligonucleotide primers for            RT-PCR screening with mRNA from tissues such as but not            exclusively, the hearth and the brain.

Production of recombinant NL-enzymes

-   -   As shown in the present work, recombinant active NL-enzymes can        be obtained by expression of NL-cDNAs in mammalian cells. From        past experience with neprilysin, another member of the family        (Devault et al., 1988; Fossiez et al., 1992; Ellefsen et al.,        submitted), expression can also be performed in other expression        systems after cloning of NL-cDNAs in appropriate expression        vectors. These expression systems may include but not        exclusively the baculovirus/insect cells or larvae system and        the Pichia pastoris-based yeast system. Production of        recombinant NL-enzymes includes the production of naturally        occurring membrane bound or soluble forms of the proteins or        genetically engineered soluble forms of the enzymes. The latter        can be obtained by substituting the cytosolic and trans-membrane        domain by a cleavable signal peptide such as that of        proopiomelanocortin, but not exclusively, as done previously        (Lemay et al., 1989) or by transforming by genetic manipulations        the non-cleavable signal peptide membrane anchor domain into a        cleavable signal peptide, as done previously (Lemire et        al., 1997) or by fusion of the ectodomain of NL-enzymes to the        amino-terminal domain (from the initiator methionine to amino        acid residue 300) of naturally occurring soluble NLs such as,        but not exclusively, NL-1 as done in this work. These        recombinant NLs could be used to:        -   find a substrate. A substrate can be identified using one of            the following.        -   Screening of existing bioactive peptides. Peptides are            incubated in the presence of NL-enzymes and subsequently            analysed by HPLC for degradation. Degradation is observed by            disappearance of the peak of substrate and the appearance of            peaks of products;        -   Screening phage libraries specifically designed for the            purpose (phage display library). Each phage expresses at its            surface, as part of its coat protein, a random peptide            sequence preceded by a peptide sequence recognisable by an            antibody or any other sequence-recognizing agent. This            latter sequence serves to attach the phage to a solid            support. Upon addition of the NL-enzyme the random sequences            that are NL substrate are cleaved, releasing the phage.            After several rounds of cleavage, the phage sequence is            determined to identify the peptide segment recognized by the            enzyme.        -   Extract of the tissue where the enzyme is expressed is            collected and prepared for chromatographic analysis (HPLC,            capillary electrophoresis or any other high resolution            separation system) by denaturing the extracted proteins with            a solvent (acetonitrile or methanol). The extract is            subjected to chromatographic separation. The same extract is            incubated with the enzyme for a period sufficient to observe            a difference between the 2 chromatograms. The regions with            the identified changes are collected and subjected to mass            spectrometric analysis to determine the peptide            compositions.        -   Small peptide libraries are prepared with a fluorophore at            one extremity and a quencher group at the other (Meldal et            al Methods in molecular biology 1998,87). The substrate can            be identified using a strategy described in Apletalina et al            (JBC (1998)273, 41, 26589–95). For each hexapeptide library,            the identity of one residue at one position remains constant            while the rest is randomized (for a total of 6*20=120            individual libraries). Each library is made-up of 3.2            million different members and is identified both by the            position of the constant residue along the hexapeptide, and            its identity. The NL-enzyme is added to each library and the            fluorescence is recorded. The data is organized to identify            the libraries producing the most fluorescence for each            position along the hexapeptide. This arrangement suggests            the identity of important residues at each position along            the hexapeptide. Hexapeptide representing the best            suggestions are prepared and tested in a similar fashion.            From this set, the hexapeptide with the best fluorescence is            selected.        -   set up enzymatic assays. An enzymatic assay consists in the            addition of the above-identified substrate to the enzyme in            constant conditions of pH, salts, temperature and time. The            resulting solution is assayed for the hydrolysed peptide or            for the intact peptide. This assay can be realized with            specific antibodies, HPLC or, when self-quenched            fluorescence tagged peptides are used (Meldal et al), by the            appearance of fluorescence. The enzyme may be in solution or            attached to a solid substrate;        -   identify inhibitors. Inhibitors can be identified from            synthetic libraries, biota extracts and from rationally            designed inhibitors using X-ray crystallography and            substituent activity relationships. Each molecule or extract            fraction is tested for inhibitory activity using the            enzymatic test described above. The molecule responsible for            the largest inhibition is further tested to determine its            pharmacological and toxicological properties following known            procedures. The inhibitor with the best distribution,            pharmacological action combined with low toxicity will be            selected for drug manufacturing. Pharmaceutically acceptable            formulation of the inhibitor or its acceptable salt will be            prepared by mixing with known excipients to produce tablets,            capsules or injectable solutions. Between 1 and 500 mg of            the drug is administered to the patients;        -   inject the native or soluble purified NL-enzymes into            subjects. In the case of disease or pathologies caused by a            lack or decrease in NL activity, the purified NL could be            injected intravenously or otherwise in patients.            Alternatively, immobilized NL-enzymes could be introduced at            the site of orthopedic surgery or implantation of devices in            bones or dental tissues.

Secretion of foreign proteins and peptides

As shown in the present work, the amino-terminal domain of NL-1 (fromthe initiator methionine to the furin site) can be used to promote thesecretion of a foreign protein (in this case the ectodomain of NL-3 andβ-endorphin).

The amino-terminal domain of NL-1 but also of other naturally occurringsoluble

-   -   NL-enzymes could be used to:        -   promote production and secretion of foreign proteins. This            can be achieved by genetically fusing sequences coding for            said foreign proteins downstream from and in phase with the            amino-terminal of NL-1. These chimeric constructs could be            introduced with the help of appropriate vectors in any of            the expression systems mentioned above for protein            production and secretion;        -   promote production and secretion of bioactive peptides.            Sequences encoding small bioactive peptides such as but not            exclusively β-endorphin, the enkephalins, substance P,            atrial natriuretic peptide (ANF) and osteostatine, could be            fused immediately downstream and in phase the furin site of            NL-1. These DNA constructs could be used as described above            to produce bioactive peptides.        -   serve as model to design artificial (non-naturally            occurring) proteins or protein segments (protein vectors) to            promote secretion of proteins or peptides. These protein            vectors can be constructed to resemble a secreted protein.            In this case they would be assembled of an endoplasmic            reticulum signal peptide, a spacer of varying length and a            furin cleavage site to which the protein or peptide destined            for secretion can be fused. The total length of the spacer,            furin cleavage site and protein or peptide destined for            secretion must be at least 70 amino acid residues.            Alternatively, such protein vectors could be assembled to            resemble a type II membrane protein. In this case they would            comprise from the amino to the carboxy-terminus a cytosolic            domain of varying length, a transmembrane domain that also            acts as a signal peptide, an extracellular segment of            varying length and a furin cleavage site to which the            protein or peptide destined for secretion can be fused. The            total length of the extracellular segment, furin cleavage            site and protein or peptide destined for secretion must be            at least 70 amino acid residues.            Therapeutic applications of NL-enzymes

The inappropriate processing of endogenous peptides causes severaldiseases. The inappropriate processing may result from pathologicconcentration of the enzyme itself, its substrate or other elements ofthe biochemical machinery downstream from the controlling enzyme. Inthis context it is possible to help the patient by managing the activityof the controlling enzyme.

-   -   -   NL-enzymes have been localized to the brain and may be            involved in the improper processing of β-amyloid precursor.            Inhibitions of this process by drugs prepared as above, will            help patients with Alzheimer disease as well as other            patient suffering from diseases caused by plaque formation;        -   NL-enzymes may be involved in the improper processing of            other peptides involved in neurological diseases, pain or            psychiatric disorders. Appropriately designed inhibitors            will help in the management of such diseases;        -   NL-1 is found in testis and is associated with spermatozoid            maturation. Peptides improperly processed by the enzyme may            lead to infertility. The addition of NL-1 ex-vivo to seminal            liquid or immature spermatozoids taken directly from testis            during an in-vitro fertilization procedure will increase            fertility. Conversely, the use of a small-molecule inhibitor            or removal of NL-1 with an antibody could increase fertility            during an in-vitro fertilization procedure. The            administration of a NL-1 inhibitor may increase or decrease            the fertility potential. This inhibitor is formulated and            administered as described above.        -   NL-3 is found in ovaries and may be involved in the            processing of a peptide involved in the maturation of eggs.            The addition of NL-3 ex-vivo to immature eggs taken directly            from ovaries during an in-vitro fertilization procedure will            increase fertility. Conversely, the use of a small-molecule            inhibitor or removal of NL-3 with an antibody could increase            fertility during an in-vitro fertilization procedure. This            inhibitor is formulated and administered as described above;        -   NL-3 is found in bones. The improper processing of peptides            by the enzyme may result in bone disease or abnormal            phosphate metabolism. Administration of an inhibitor, as            described above, will allow the disease management.

TABLE I Extend of amino acid sequence identity between members of theNEP-like family hECE- hECE- sNL- hNL- hNL- hNEP hPEX 1A 2 hKELL 1 2 3hNEP 100* hPEX 35 100 hECE-1A 39 38 100 hECE-2 36 37 62 100 hKELL 23 2430 31 100 sNL-1 55 39 39 39 26 100 hNL-2 54 39 39 39 26 77 100 hNL-3 3532 37 37 28 36 34 100 *percentage of sequence identity

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1. An isolated nucleic acid comprising a nucleotide sequence encoding ametallopeptidase having at least about 95% amino acid sequence identitywith the complete amino acid sequence of SEQ ID NO:
 13. 2. A recombinantvector comprising the isolated nucleotide sequence of claim
 1. 3. Anisolated host cell transformed with the vector of claim
 2. 4. Anisolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:12.
 5. A recombinant vector comprising the isolated nucleotide sequenceof claim
 4. 6. An isolated host cell transformed with the vector ofclaim 5.